ProjectSummary/Abstract The emergence of antibiotic resistance has created a global dilemma for the need to discover new antibacterial lead agents. As the majority of antibiotics are derived from nature, and in particularfromterrestrialsoilbacteria,inrecentyearsmarinebacteriahavebeenestablishedas promising sources of antibacterial compounds. An underlying theme associated with many marine microbial antibiotics involves the use of aromatic polyketide frameworks that have undergone extensive oxidative tailoring reactions catalyzed by halogenase and oxygenase biosynthetic enzymes. In this application, we propose a multidisciplinary project involving heterologous biosynthesis, mechanistic enzymology, atomic resolution protein X-ray crystallography, chemoenzymatic synthesis, and genetic engineering to understand the molecular basis of polyketide diversification in a series of marine bacterial antibacterial agents withpromisingbiologicalproperties.Toaccomplishthebroadgoalsoutlinedinthisapplication, weproposefourspecificaims.First,weplantofunctionallyandstructurallycharacterizediverse meroterpenoid V-dependent chloroperoxidases and their catalytic properties in promoting antimicrobial chemical diversity. Second, we will discover, characterize, and engineer biosynthetic pathways for structural diversification of halogenated pyrrole containing bioactive natural products. Third, we aim to functionally characterize the biosynthesis of the thiolactomycinpolyketideantibioticsandtheirnovelsulfurinsertionenzymology.Andfourth,we will functionally characterize the salinamide ether bridge forming enzymes and design new derivativesforbiologicalevaluation.